The present invention relates to the production and use of transgenic animal models and includes methods of modulating p21 phenotypes in a ras oncogene background. More particularly, the invention relates to the production and use of transgenic animals having decreased or null p21 expression in combination with constitutively activated ras expression, which are characterized by early and frequent tumorigenesis, and which provide improved models for the study of tumorigenesis and screening of anti-cancer agents and treatments.
The ras oncogenes are frequently activated by point mutations or overexpression in human tumors. About 30% of all human tumors are associated with ras mutations and up to 95% of human pancreatic cancers contain K-ras mutations (Barbacid, 1990; Bos, 1989). Ras proteins are believed to mediate their oncogenic effect, in part, by dysregulating the cell cycle machinery (Downward, 1997; Ewen, 2000; Kerkhoff and Rapp, 1998). For instance, activated Ras, acting through the Raf/Mek/Erk kinase pathway, increases cyclin D1 expression and shortens the G1 phase of the cell cycle (Hitomi and Stacey, 1999; Liu et al., 1995). Moreover, Raf kinase was shown to interact with and regulate the activity of cdc25A, a phosphatase involved in the regulation of cdc2, an essential G2/M kinase (Xia et al., 1999). Unlike NIH3T3 cells, which have lost cyclin dependent kinase inhibitors (CKI) such as p16 or p19, activated Ras alone does not transform primary murine fibroblasts, but requires a cooperating oncogene (Franza et al., 1986; Hirakawa and Ruley, 1988; Parada et al., 1984; Yancopoulos et al., 1985). In fact, by itself, activated Ras causes growth arrest in these cells. From recent work, it is now apparent that this growth inhibitory effect of a strong Ras signal is due to the ability of the Raf/Mek/Erk pathway to induce expression of the CKI p21WAF1/CIP1 (hereafter referred to as p21). A strong indication that the cell cycle arrest induced by a high intensity Ras/Raf signal is mediated by a high p21 level comes from the observation that in p21-deficient fibroblasts these signals do not lead to cell cycle arrest (Olson et al., 1998). These studies point to p21 as being one of the targets of the Ras/Raf signaling pathway. It is possible that the apparent ability of oncogenic Ras or Raf to induce the expression of p21 is a protective or stress response of the cell to receiving a strong Ras/Raf signal at an inappropriate stage in the cell cycle. p21 is induced in response to a broad spectrum of cellular stresses, thus allowing the cell to halt cell cycle progression. In normal human cells, p21 exists in a quaternary complex with a cyclin, a CDK, and the proliferating cell nuclear antigen (PCNA), a processivity factor of DNA polymerase xcex4 (Zhang et al., 1993). p21 modulates CDK activity, thereby affecting cell cycle progression, whereas its effect on PCNA may be important in DNA replication and/or excision repair.
Investigation of naturally arising tumors has indicated that unlike p16, deletion or mutation of the p21 gene is not common in human tumors and is not a probable mechanism of inactivation of this gene (Balbin et al., 1996; Gao et al., 1995). The involvement of p21 in tumor suppression has also been questioned, as mice lacking p21 undergo a normal development, harbor no gross alterations in their organs, and exhibit no increase in spontaneous tumor development (Brugarolas et al., 1995; Deng et al., 1995). However, even in p53-deficient mice, spontaneous tumor formation occurred only in few tissues (Jacks et al., 1994). A similar study failed to show an effect of p21 on tumors formed by ras-transformed keratinocytes (Weinberg et al., 1997).
In the study of, and development of treatments for, ras-dependent tumors, there is a need for rapid means of screening possible compounds and treatments for efficacy. While it is desirable to use whole animals for this purpose to approximate at closely as possible the response of a patient and to enhance detection of toxicity, most mammalian tumor models suffer from a drawback in the length of time that is required to perform such a screening. Thus, suitable animals must be reared and allowed or induced to produce a suitable burden of tumors, which may take many months and is therefore costly in time and materials. Therefore, animal models in which the time required to produce a suitable tumor burden is shortened would be of great significance in accelerating the development of cancer treatments and the identification of new anti-cancer drugs or lead compounds.
In the present invention described herein, the aforementioned drawback is overcome by crossing p21-deficient mice with tumor-susceptible ras transgenic mice, wherein it is demonstrated by way of non-limiting example that p21-deficiency against a ras background dramatically accelerates the onset, and increases the multiplicity and aggressiveness, of Ras-dependent tumors.